Prion-Like Domain Dysfunction in SMN1 Initiates Aberrant hnRNP Assembly, Multiple Protein Aggregation and Causing Spinal Muscular Atrophy

doi:10.21203/rs.3.rs-3341542/v1

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Prion-Like Domain Dysfunction in SMN1 Initiates Aberrant hnRNP Assembly, Multiple Protein Aggregation and Causing Spinal Muscular Atrophy

https://doi.org/10.21203/rs.3.rs-3341542/v1

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Many proteins linked to neurodegenerative diseases, such as tau, TDP-43, and alpha-synuclein, contain specialized segments known as prion-like low-complexity (LC) domains. Although inherited mutations in these proteins often occur within these domains, their exact role in neuropathology remains unclear. Spinal muscular atrophy (SMA), caused by loss of SMN proteins, is an autosomal recessive disease involving motor neuron degeneration. Herein, we found that SMN1 contains a prion-like domain in exons 6-7. Loss of the prion-like domain of SMN1 not only causes condensatopathy of gems but also disrupts the homeostasis of prion-like hnRNPs and triggers the protein aggregation of prion-like partners of SMN1, i.e., TDP-43 and PFN1, and motor neurodegeneration. Interestingly, SMN2, a near-identical gene, lacks this domain. We found that treating cells with a drug called baicalein enables SMN2 to adopt a prion-like structure, restoring phase condensation, eliminating TDP-43 and PFN1 protein aggregates, and improving SMA symptoms in models. In summary, the loss of SMN1's prion-like domain leads to a cascade of negative cellular events and motor neuron degeneration. Baicalein is the first drug identified to correct these pathologies by restoring prion-like characteristics to the affected proteins. Our findings suggest that this approach could be vital in treating a wide range of neurodegenerative diseases by stabilizing prion-like folding, normalizing phase condensation, and preventing multiple proteinopathies.

Biological sciences/Neuroscience/Molecular neuroscience

Health sciences/Neurology/Neurological disorders/Neuromuscular disease

survival motor neuron (SMN) proteins

spinal muscular atrophy (SMA)

prion-like low complexity (LC) domain

liquid-liquid phase separation

misfolded protein aggregation

gems and baicalein

A novel type of protein‒protein interaction domain, referred to as the prion-like low-complexity (LC) domain, transiently forms a homotypic or heterotypic cross-β polymeric condensed phase to perform crucial neuronal functions^{1, 2}. The prion-like LC domain was originally found to assemble membrane-less organs such as RNA granules, nucleoli, and nuclear bodies by liquid‒liquid phase separation (LLPS). Proteins with prion-like LC domains are involved in various biological processes, including DNA transcription and replication, chromatin remodeling, nuclear pore passage, signal transduction, synaptic transmission, and cytoskeleton regulation^3,4,5,6. To date, tau, TDP-43 and alpha-synuclein have been identified as prion-like LC proteins, and disease-associated mutations in the genes encoding these proteins are mainly in the prion-like LC domains. However, loss of cross-β interactions of prion-like LC domains, particularly loss of heterotypic interactions of the prion-like domains of disease-causing proteins and their prion-like interaction partners, contributes to neurodegeneration remains to be further investigated.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that affects approximately 1 in 10,000 babies born worldwide each year^7,8. SMN protein deficiency results in the gradual loss of motor neurons in the anterior horn of the spinal cord and subsequent system-wide atrophy of skeletal muscles⁹. Survival motor neuron 1 (SMN1), has been identified as the gene responsible for SMA¹⁰. Herein, we found that SMN1 contains a prion-like LC domain in exons 6-7 that drives LLPS of gems and that SMA is caused by loss of the prion-like domain of SMN1. Furthermore, we found that defects in the prion-like LC domain of SMN1 causes not only condensatorypathy but also collapse of cellular cross-β networks, i.e., aberrant prion-like assembly of RNPs, and protein aggregation of the SMN interaction partners TDP-43 and PFN1.

A nearly identical copy of SMN1, SMN2, is normally expressed in all patients with SMA. Although a small amount of full-length protein that is identical to SMN1 is produced, an exon-splicing silencer bearing a C-to-T transition in exon 7 of SMN2 in all individuals skips exon 7^{11, 12}. We found that exon 7 deletion leads to loss of SMN1 prion-like activity. Thus, it is clear that loss of a prion-like domain, such as in SMA, can cause aberrant assembly of RNPs, multiple proteinopathies and neurodegeneration. Significantly, baicalein restores the ability of the SMNΔ7 protein, which is encoded by SMN2, to adopt a prion-like conformation, effectively restoring the cellular functions of SMN1 and ameliorating SMA in human dermal fibroblasts (HDFs) and a mouse model. Therapeutic drugs, such as baicalein, can restore prion-like interactions and phase condensation and therefore may contribute to more effective treatment of neurodegenerative diseases.

Deficiency of the Prion-Like Domain of SMN1 Causes Aberrant RNP Assembly, PFN1 and TDP-43 Aggregation

Biotinylated isoxazole (b-isox), a recently identified chemical probe, specifically recognizes cross-β prion-like polymers and sequentially precipitates with proteins harboring prion-like, low-complexity (LC) or phase-separated domains, such as TDP-43, Fus and tau². To determine whether SMN is a prion-like LC protein, we initially incubated 100 mM b-isox with mes23.5 and 293T cell lysates at 4°C to chemically precipitate whole prion-like proteins and then analyzed the efficiency of SMN binding by Western blotting to assess the prion-like and phase transition potentials of SMN (Fig. 1a and Extended Data Fig. 1). Western blot analysis revealed that SMN was precipitated by b-isox (Fig. 1a). Subcellular fractionation analysis revealed that the major cross-β conformation of SMN recognized by b-isox was localized in the cytosol (Fig. 1a). SET and PFN1 were used as fractionation controls (Fig. 1a).

To locate the prion-like domain of SMN, we examined the capability of full-length SMN (SMN-FL) and a panel of exon-deleted SMN constructs to form gems, i.e., their LLPS capability. A map of the SMN variants and the results are shown in Fig. 1b. Only fragments containing the region encoding exons 6-7 formed visible granules (Fig. 1b, arrowhead). The dynamics of mCherry-SMN_exon6_-₇granules in living cells were further analyzed by time-lapse microscopy, and the trajectory of mCherry-SMN_exon6_-₇granules is shown in Fig. 1c. Most of the mCherry-SMN_exon6_-₇granules traveled within a limited region (Fig. 1c), and two independent mCherry-SMN_exon6_-₇granules occasionally underwent fission and fusion (Fig. 1d, arrowheads). The average speed of mCherry-SMN_exon6_-₇granules was ~0.8 µm/s. As the prion-like domain of SMN is located in exons 6-7, we inferred that the SMNΔ7 protein, which is encoded by SMN2, is an inactive prion-like SMN protein.

In addition to engaging in homotypic interactions to assemble membrane-less organelles, such as the gem described above, prion-like LC domains also engage in heterotypic cross-β interactions. We thus conducted a detailed examination of SMN-associated prion-like proteins in SMA models. First, we assessed the global changes in prion-like LC protein expression in SMA. Spinal cord lysates were collected from type I SMA-like mice and control littermates, and prion-like LC proteins were precipitated by b-isox and identified through LC-MS/MS¹³. Differential b-isox-bound proteins were subsequently analyzed by the DAVID Functional Annotation Bioinformatics Analysis (https://david.ncifcrf.gov/tools.jsp) database, and the enriched Gene Ontology (GO) terms were identified. In SMA mice, according to GO enrichment analysis (FDR-adjusted P £ 4.95E-5, Fig. 1e), the expression of b-isox-precipitated proteins involved in RNA binding and RNP assembly was reduced, indicating a key role for SMN in the prion-like assembly of RNPs¹⁴. Consistent with the results of GO enrichment analysis, 15 out of 48 downregulated proteins were RNA-binding proteins (Fig. 1e).

Next, immunofluorescence staining revealed that an SMN prion-like interaction partner, PFN1, formed cytosolic aggregates in SMA patient-derived human dermal fibroblasts (HDFs) (Fig. 1f). PFN1 is a known ALS-causing protein; it becomes misfolded and forms cytosolic misfolded aggregates in patients with ALS^15,16,17. To confirm that deficiency of one prion-like protein abolishes the prion-like folding of its interaction partners, causing them to form aggregates, we examined the localization of another prion-like interaction partner of SMN1, TDP-43, in spinal cord motor neurons in SMA mice (Fig. 1g). TDP-43 is a key molecule in ALS, as misfolded TDP-43 aggregates have been observed in ~80% of ALS patients¹⁸. Indeed, the results of TDP-43 immunofluorescence showed TDP-43 mislocalization in the cytosol (arrowhead) and deposition on the nuclear envelope (arrow) in the motor neurons of SMA mice. Of note, the aggregation of ALS-causing proteins in SMA has never been reported. These findings related to PFN1 and TDP-43 reveal that SMA and ALS share a common etiology.

Finally, we treated healthy HDFs with b-isox to interfere with the cross-β interactions of prion-like LC proteins and found that the number of gems was significantly reduced. Conversely, the number of PFN1 aggregates increased in a dose-dependent manner, confirming that disrupting prion-like cross-β interactions can lead to protein aggregates (Fig. 1h).

Baicalein-Mediated Conformational Alteration of SMN2 Triggers Phase Separation and Endows SMN2 with SMN1 Prion-Like Functions

In searching for an activator to restore SMN cross-β interactions, we found that baicalein normalizes the assembly of gems in type III SMA patient-derived HDFs by screening a chemical library (Fig. 2a)¹⁹. On the other hand, we noticed that after treating SMA patient-derived HDFs with baicalein, TDP-43 aggregates disappeared (Fig. 2b). Statistical analysis of data related to the effect of baicalein on the formation of gems is shown in Fig. 2c. As SMA patient-derived HDFs express only the SMN2 protein, we speculated that baicalein restores the formation of gems in SMA patient-derived HDFs by altering the conformation of the nonfunctional prion-like C-terminus of SMN2. We used the b-isox precipitation assay to assess the cross-β conformation of SMN2 in vivo and found that the cross-β conformation of SMN2 in baicalein-treated SMA patient-derived HDFs was the same as that of SMN-FL (Fig. 2d). These results suggest that baicalein may endow the nonfunctional SMN2 C-terminus with the ability to adopt a prion-like conformation.

We next assessed whether these SMN2 proteins with baicalein-induced restoration of prion-like activity can perform the unique cellular functions of SMN1. We measured the length of axons of neurons transfected with SMNΔ7 in the presence or absence of baicalein, as neurons transfected with SMNΔ7 extend significantly shorter neurites than those transfected with SMN-FL^{20, 21}. Indeed, baicalein increased the length of axons from NSC34 cells expressing SMNΔ7 by approximately 2 fold (Fig. 2e); the results of statistical analysis are shown in Fig. 2f. As the protein half-life of SMNΔ7 is two-fold shorter than that of SMN-FL in cells, we examined the stability of the SMNΔ7 protein in the presence of baicalein and found that baicalein significantly increased the stability of the SMNΔ7 protein (Fig. 2g, arrow)²². Additionally, as PFN1 is known to effectively interact with SMN but not SMNΔ7, we analyzed the interactions of SMNΔ7 and PFN1 in baicalein-treated cells by coimmunoprecipitation (Fig. 2h)^15,16. The results showed that in the presence of baicalein, the interaction between PFN1 and SMNΔ7 was significantly increased (Fig. 2h, arrowhead).

To comprehensively identify the pharmacological targets of baicalein in SMA, we collected protein lysates from NSC34 cells treated with or without baicalein and then precipitated the proteins with b-isox. The proteins precipitated by b-isox were then identified by LC‒MS/MS. Differential protein expression was subsequently analyzed by the DAVID database, and enriched GO terms were identified. Importantly, in baicalein-treated NSC34 cells, the top 5 upregulated GO terms were mostly related to the cellular functions of Smn, including Sm core formation, SnRNP assembly, RNA binding, and RNA splicing (FDR adjusted P £ 1.09E-9, Fig. 2i). Among the 71 proteins whose expression was regulated by baicalein, 26 were directly associated with Smn or involved in Smn complexes (Fig. 2i)²³. Together with the results in Figure 2, these findings suggest that baicalein endows SMN2 with prion-like bioactivity, allowing it to behave similarly to SMN1.

Baicalein Improves Survival and Rescues Gem Assembly and Motor Neuron Function in SMA Mice

To determine whether the in vitro findings could be recapitulated in vivo, we administered baicalein (13.6 mg/kg/d) or DMSO (control) to late-onset type III SMA model mice daily by intraperitoneal injection¹⁹. After 4 weeks of treatment, histological analysis of lumbar spinal sections from SMA mice confirmed an increase in the ChAT-positive motor neuron number (Fig. 3a, P=0.0435) and recovery of nuclear gem formation (Fig. 3a, bottom, P=0.0063, labeled by arrowheads in Fig. 3a).

We found that SMN protein levels but not SMN mRNA levels were increased in various tissues from baicalein-treated SMA mice compared with those from control SMA mice (Fig. 3b) (Extended Data Fig. 2), confirming that baicalein increases the protein stability of SMND7, consistent with the data shown in Fig. 2g. Beginning at 7 months of age, we treated type III SMA mice with baicalein for four months, which improved mouse survival (P=0.048) (Fig. 3c). A reduction in the evoked compound muscle action potential (CMAP) amplitude upon sciatic nerve stimulation was observed in control SMA mice (Fig. 3d, left, P=0.0201), but this phenomenon was abolished in baicalein-treated SMA mice (Fig. 3d, right, P=0.3326). Baicalein-treated SMA mice showed better motor performance than control mice in both the fixed-speed and accelerating rotarod tests, as determined by measuring the latency to fall (Fig. 3e and f). Fluorogold retrograde tracing of spinal motor neurons revealed a higher motor neuron density within the anterior horn (Fig. 3g, left, P=0.0114) and more large a-motor neurons (Fig. 3g, right, P=0.0037) in baicalein-treated mice than in control SMA mice. Significantly, baicalein treatment increased the axonal innervation of neuromuscular junctions (NMJs) in the hamstring muscles of SMA mice (Fig. 3h, P=0.04).

Next, we administered baicalein (13.6 mg/kg/d) or DMSO (control) to early-onset type I SMA model mice daily by intraperitoneal injection beginning at birth. We expected that the therapeutic efficacy of baicalein on type I SMA mice was weaker, as SMN2 protein levels are lower in type I SMA than in type III SMA. After baicalein treatment, the motor function of SMA mice, including the righting time, tube score, and tilting score, was improved on the 6th postnatal day (P<0.05), and the results were similar to those for heterozygous littermates treated with or without baicalein (Extended Data Fig. 3). On the 8th postnatal day, the motor function of baicalein-treated SMA mice was better than that of untreated SMA mice in the tube test (P=0.009) but not in the turnover test (P=0.065) or negative geotaxis test (P=0.58) (Extended Data Fig. 2). However, baicalein-treated type I SMA mice showed lifespans (8.8±0.4 vs. 7.9±0.2 days; P=0.13) and body weight gains that were similar to those of control SMA mice. The weaker effects of baicalein treatment on type I SMA mice than on type I SMA mice supports that baicalein targets to SMN2, endowing it with prion-like activity to perform the prion-like functions of SMN1 in vivo.

The strong correlation between the self-association of the SMN protein and disease severity suggests that SMN self-oligomerization is a prerequisite for essentially all SMN functions^{24, 25}. Here, we discovered that the self-oligomerization of SMN is driven by an intrinsic prion-like conformation and identified SMA as a novel conformational disease. Unlike the various conformational neurodegenerative disease-causing proteins, such as TDP-43 and tau, which become misfolded and form inclusions, misfolded SMN1 and exon 7-deleted SMN2 simply abolish prion-like cellular functions and leads to rapid degradation¹⁸. We thus propose an as yet unidentified group of neurodegenerative diseases in which prion-like proteins are deleted or rapidly degraded. Although these disease-causing proteins, such as SMN, do not form aggregates, loss of prion-like cross-β interactions leads to aggregation of misfolded prion-like interaction partners and consequentially induces proteinopathies.

Based on the unique role of prion-like conformations in motor neurons, we propose a type of therapeutic strategy for SMA, i.e., “conformation-based therapies”, in which SMA disease-causing mutants and SMNΔ7 are converted into prion-like folded proteins (Fig. 3i). Baicalein endows SMN mutants and SMN2 with prion-like activity, subsequently triggering LLPS of SMN2 to form gems; increasing prion-like interactions, i.e., the SMNΔ7-PFN1 interaction; reducing the protein degradation of SMN; and improving motor neuron survival and motor function in SMA models. We term these baicalein-reassembled prion-like conformers of SMN2 prion-like iso-conformers. Our study presents a groundbreaking perspective on the therapeutic landscape of neurodegenerative disorders, elucidating the critical significance and indispensable nature of restoring prion-like interactions and phase condensation. It stands as a pioneering contribution, shedding light on a novel and essential avenue for addressing these complex diseases.

Acknowledgments

We thank the staff at the Image Core at Academia Sinica and the Core Labs at the Department of Medical Research, National Taiwan University Hospital for their assistance with the confocal microscopy and technical support. The SMN constructs were kindly gifted by Dr. Li-Hung at IMB, Academia Sinica. We dedicate this work to Dr. Hung-Li and commemorate and thank him for inspiring our academic careers. This research was funded by Garage Brain Science (ND-002), YEEFAN Med Inc. (SMA-01), and was supported by the Ministry of Science and Technology in Taiwan to Dr. L.-K. Tsai (108-2314-B-002-082-MY3).

Conflict of Interest Statement

H.-Y. Chang, C.-H. Ting and I.-F Wang are financially compensated as employees of Garage Brain Science Co., Ltd. in Taiwan and YEEFAN MED INC. in the US.

Authors’ Contributions:

H.-Y. Chang and I.-F. Wang characterized the prion-like properties of SMN1. C.-H. Ting analyzed axonal outgrowth and gem bodies in patient-derived HDFs and performed time-lapse recording. C.-H. Ting, H.-J. Lai, C.-L. Chen and L.-K. Tsai performed the pharmacological analysis of baicalein in SMA mice. C.-H. Ting, L.-K. Tsai, H.-Y. Chang, and I.-F. Wang wrote the manuscript, which was approved by all coauthors.

Cell Culture and Drug Treatment

Human dermal fibroblasts (HDFs) were cultured in medium 106 (Gibco®, Thermo Fisher Scientific) with low serum growth supplement (LSGS) (Gibco®, Thermo Fisher Scientific) and 1% penicillin-streptomycin (Gibco®, Thermo Fisher Scientific) as previously described¹⁹. To determine the effects of compounds on the LLPS of prion-like proteins, the cells were treated with baicalein or b-isox for 24 or 48 h at the indicated concentrations (5, 10, 25, or 50 μM). Transfections were performed using Lipofectamine^TM 2000 or Lipofectamine^TM 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, cells were seeded into the wells of a 96-well plate (7.5´10³ cells for viability assays), a 12-well plate (1.5´10⁵ for immunocytochemistry (ICC) studies) or a 6-well plate (5´10⁵ for time-lapse studies) and cultured overnight. Cells transfected with mCherry-SMND7 expression plasmids were assayed for neurite outgrowth at 48 h posttransfection. Baicalein (50 mM) was added at 24 h posttransfection, and neurite outgrowth was determined 24 h after drug addition.

Subcellular Fractionation and Western blotting

Cultured 293T and mes23.5 cells were harvested using a subcellular protein fractionation kit (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Cell lysates were separated into five different cellular compartments. The fractionated proteins were used for Western blotting, as previously described²⁸. The SMN and mCherry antibodies were kind gifts from Hung-Li. The profilin1 antibody was purchased from Proteintech (11680-1-AP).

b-isox Precipitation

Briefly, 293T cells were harvested and lysed with RIPA buffer. The protein concentration was adjusted to 1 to 10 mg/mL, and 10 mM b-isox was added to the cell lysate at a final concentration of 100 to 200 mM. The mixtures were then incubated at 4°C for 60 min and centrifuged at 15000 rpm for 15 min at 4°C, and the supernatant was discarded. All of the mixtures were subjected to SDS-PAGE and Western blotting.

Mice

A mouse model of SMA was produced by deleting exon 7 of the Smn gene and knocking in the human SMN2 gene (Smn^-^/^-SMN2⁺^/^-). We were able to generate a variant that exhibited more severe disease symptomatology by backcrossing to obtain a more homogenous genetic background with two copies of the SMN2 transgene (Smn^-^/^-SMN2⁺^/^-) (severe SMA model). We also generated a model of mild SMA, which carried four copies of the SMN2 transgene (Smn^-^/^-SMN2⁺^/⁺)^. Both severe and mild SMA mice received daily intraperitoneal injections of baicalein (13.6 mg/kg/d in DMSO) or DMSO alone (control) from birth until death (severe SMA mice) or from 7 to 11 months of age (mild SMA mice). The severe SMA mice were then subjected to motor function tests and survival analysis. Mild SMA mice were subjected to motor function tests, survival analysis, electrophysiological studies, and pathological studies. Another group of mild SMA mice received the same dose of baicalein or DMSO at 4 months old for 2 weeks for Western blot and quantitative PCR analyses. Housing and husbandry were maintained with standard procedures in the NTUCM Laboratory Animal Center. All animal procedures in this study were approved by the Institutional Animal Care and Use Committee (protocol #20190314).

Motor Function Tests

For mild SMA mice, both fixed-speed (20 rpm for 5 min) and accelerating (from 4-40 rpm in 5 min) rotarod test protocols were conducted to evaluate motor function every 2 weeks, as previously described¹⁹. Mice were placed in a neutral position on the stationary 3-cm diameter cylinder of the rotarod apparatus (Singa RT-01), and the time to fall was recorded when each mouse fell off the rod. For severe SMA mice, the turnover, tube and negative geotaxis tests were conducted at 6 and 8 days old, as previously described¹³. In the turnover test, the time required for a mouse to right itself and place all four paws on the ground from a prone position was recorded (the cutoff time was 60 sec). The tube test was used to determine hind limb strength according to the posture of the hind limbs and the tail; scores ranged from 0 (the worst) to 4 (the best). In the negative geotaxis test, mice were placed on a 45° incline with their head pointing downward. The responses (turning and climbing) were scored from 0 (worst) to 4 (best).

Retrograde Tracing, Immunocytochemistry, and Immunohistochemistry

For retrograde motor neuron tracing, fluorogold dye (Fluorochrome, LLC) was injected into the bilateral gastrocnemius and tibialis anterior muscles of mild SMA mice (2 μL for each muscle) 3 days before sacrifice. For immunocytochemistry, HDFs were fixed and blocked as previously described¹⁹. Gem number counts and SMN nuclear localization in fibroblasts were confirmed using DAPI counterstain. For immunohistochemistry, lumbar spinal cords and hamstring muscles from mild SMA mice were fixed with 4% paraformaldehyde (PFA) and dehydrated in a series of sucrose solutions. Lumbar spinal cord cryosections (10 μm) and hamstring muscle cryosections (15 mm) were blocked, permeabilized, and incubated with the appropriate antibodies as previously described^{29, 30}. Images were obtained using a Zeiss LSM880 microscope (Carl Zeiss) with a 10´ or 20´ objective lens (plan-apochromat 20´/0.8 M27 and plan-apochromat 40´/1.3, oil, DIC M27). The mean number of motor neurons per anterior horn, nuclear gem counts per motor neuron, and percentage of axonal innervation at the NMJ were calculated for each animal. The primary antibodies included anti-SMN, anti-ChAT (EMD Millipore, #AB144P), anti-neurofilament-H 200 kDa (EMD Millipore, #AB1989), anti-synaptophysin (Abcam, #ab32127), and α-bungarotoxin Alexa Fluor 555 conjugate (Thermo Fisher Scientific, #B35451).

Electrophysiological Study

The mice were anesthetized with Zoletil (20 mg/kg, ip). The skin in the right hind limb was shaved and adorned with surface nonpolarizable Ag/AgCl electrodes (Ambu^Ò Blue Sensor NF, Ambu A/S, Denmark), which were trimmed to approximately 0.5cm ´ 0.5cm over the gastrocnemius muscle. An invasive needle electrode was placed near the sciatic notch for stimulation, and the ground was placed on the tail with an invasive needle electrode. The recording was performed with a Nicolet EDX System and Synergy interface (Natus Medical Incorporated, USA). The stimulation was increased gradually to obtain supramaximal stimulation and a stable CMAP amplitude.

Time-lapse Study

Briefly, 293T cells were first seeded on glass slides in 6-well culture plates and then transfected with mCherry-tagged plasmids. After 48 h, a glass slide was placed into a perfusion, open, and closed (POC) chamber (Pecon) and into an incubator containing a spinning disk confocal microscope (Axio-observer. Z1/7, Zesis). Images were acquired under a 63´ objective lens (plan-apochromat 63´/1.40, oil, DIC, M27) using the mCherry (EX 572/35, EM 632/60) filter set for 2 min at appropriate time intervals. Analysis was conducted using the integrated ZEN (v.3.1) imaging program and Imaris software (v.9.6.0, Bitplane).

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Prion-Like Domain Dysfunction in SMN1 Initiates Aberrant hnRNP Assembly, Multiple Protein Aggregation and Causing Spinal Muscular Atrophy

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